Bordetella pertussis, is a worldwide pathogen which causes whooping cough in children and persistent bronchitis in adults who probably serve as the natural reservoir. Even though there is a vaccine program in this country, there are 5000 reported cases of whooping cough and an estimated 50,000 unreported cases, an incidence of disease on par with bacterial meningitis. The high reactogenicity of the diphtheria-pertussis-tetanus vaccine, due to the pertussis component, underscores the need to develop an effective subcomponent vaccine. In order to succeed in this effort, a thorough understanding of the survival strategy of the bacteria within the host environment is of paramount importance. B. pertussis has developed a survival strategy that relies on the coordinate regulation of the expression of virulence factors specifically designed to facilitate attachment thus establishing the bacteria within the host and to thwart host defenses. These virulence factors, the candidates for subcomponent vaccines, are organized into the vir regulon controlled by a single genetic locus, bvg. Coordinate regulation of the vir regulon by bvg produces virulent and avirulent phenotypes (phenotypic modulation) of this bacteria in response to the environmental signals. Well defined media components control this expression in culture, while little is known about the in vivo stimulus for phenotypic modulation. At least two different niches in the host have been identified for B. pertussis, extracellular, on the ciliated epithelium and intracellular, within phagocytes, thus heightening interest in phenotypic modulation of these virulence factors. The focus of this proposal first addresses the mechanism of coordinate regulation of the vir regulon in B. pertussis, using as a paradigm the regulatory circuit controlling the expression of the extracellular adenylate cyclase toxin (ACT), a member of this regulon and a major virulence determinant. We propose that multiple regulatory elements participate in this circuit. First we will identify these elements by transposon mutagenesis and molecular cloning. Second, the target for bvg regulation within the ACT operon will be defined by in vivo DNA footprinting and deletion, insertion and site directed mutagenesis of a 260 bp AT rich region which we have identified as containing the site for regulation. Third, the in vivo activity of this regulatory circuit will be evaluated, in this instance using bacterial invasion and survival within human macrophages as an experimental model. This will be the first time that a kinetic and temporal profile of the rapid switching by a bacterial virulence regulator will be detectable in various in vivo environments. Finally, an outline of research is presented which will biochemically and genetically characterize the role of a newly identified protein (CyaC) absolutely required for the invasive, hemolytic and probably immunogenic properties of the ACT.